Preparing for a big event takes a lot of work; whether it be the holidays or developing a brain’s epigenome, keeping a storage box ready to go for the situation just makes sense. In their latest publication, the lab of Janine LaSalle at the University of California – Davis unwrap the epigenetic storage box of neurodevelopment. In this treasure chest, they find some interesting similarities and synergies between the effects of copy number variations (CNVs) and environmental exposure on the DNA methylation landscape of Autism Spectrum Disorders (ASD).
The talented team examined one of the most common CNVs associated with ASD: Maternal duplication of 15q11.2-q13.3 (Dup15q). This region encodes the paternally imprinted, and thus maternally expressed, ubiquitin ligase UBE3A. Given the complexities of autism, they also examined the role of environmental exposures. Their poison of choice was a persistent organic pollutant known as non-dioxin-like polychlorinated biphenyl (PCB 95). PCB 95 is a developmental neurotoxin that has been banned, but still lingers in our landfills, food supply, and tissues.
LaSalle shares that “This work was based on our previous discovery of PCBs in Dup15 brains. Now, we were able to dig deep and see exactly where the genome-wide hypomethylation was located. That was actually the big surprise to me. I expected to see it over repetitive regions and the duplications, not the specific genes found.”
Using Brains for Discovery
The team used whole-genome bisulfite sequencing (WGBS) to examine 41 postmortem human brain cortical samples. Their main comparison was between individuals with Dup15q and age/sex matched controls; however, they also compared to individuals with idiopathic autism and Down syndrome as an additional level of control. Here’s what they found in Dup15q brains:
- There’s global hypomethylation, which confirms past results.
- Getting specific, there are 2,575 differentially methylated regions (DMRs), which belong to 975 genes.
- The 628 hypomethylated genes are enriched for brain functions, specifically the synaptic signaling critical to communication between neurons.
Cell Culture Confirmation
The group then turned to a SH-SY5Y cell culture model since those cells are neuron-like. They used an engineered Dup15q line and compared it to standard SH-SY5Y cells while assessing the effect of PCB 95 in both the short and long-term.
- By using the read counts from their WGBS data, they noticed that not only is the engineered 15q duplication present, but upon long-term culture and even without PCB 95, another CNV arises: Dup22q. This could be due to long-term consequences of genome instability caused by the genome-wide hypomethylation.
- Principal component analysis of the WGBS data revealed that the duplications have a much stronger effect on methylation than the long-term PCB 95 exposure.
- The team then looked for differences in partially methylated domains (PMDs), which are large-scale genomic structures of <70% methylation that are found in tissues of early development. By focusing in on hypomethylated regions, they found that 65% of altered genes were altered similarly by either PCB 95 exposure or Dup15q on their own, while 15% of the genes were only affected by the combination of the two risk factors. These genes were all enriched for synaptic signaling related functions.
- 83 genes overlapped between the methylation analysis of brains and cell culture. Those genes were also enriched for synaptic signaling and autism risk.
- By examining the expression of a select set of 40 hypomethylated genes using the Fluidigm Biomark microfluidic qPCR chip, they found most genes showed increased expression in the short-term and decreased expression in the long-term.
Co-first author Keith Dunaway shares, “In order to understand the WGBS data, I had to write my own tools. This is the first time (to our knowledge) that a cross DMR and PMD analysis was published. The long-term culture chr22 duplication would normally have gone unnoticed in most experiments. The only reason I found it was because we were wondering exactly how much of chr15 was duplicated in the cell line. Then, I looked at all of the other chromosomes out of due diligence. It wasn’t too surprising that chromosomal duplications had a larger impact on global methylation than PCB exposure. However, it was surprising that 15q duplication had a large impact on global methylation while Downs Syndrome (chr21 duplication) had none. This implies that the region duplicated had more impact on methylation than the size of the duplication.”
LaSalle continues, “I was also surprised about the overlap of genes, an interaction between the duplication and PCB 95 was expected, but I didn’t expect such a large overlap in genes for each condition independently. This shows that there is a subset of genes that are epigenetically susceptible, which function in cell membranes and neuronal synapses. These same genes are going to come back again and again. Even in an idiopathic autism brain, there are common epigenetic signatures that are going to keep coming back to genes in common pathways.”
Culturing A Mechanism
The team finally went in for some mechanistic insight by using western blots to examine UBE3A, its nuclear target RING1B, and resulting histone post-translational modifications. Co-first author M. Saharul Islam shares, “RING1B is also a E3 Ubiquitin ligase and part of polycomb repressive complex 1 (PRC1). PRC2 methylates H3K27 and PRC1 ubiquitinates the histone subunit H2A. These complexes work cooperatively to silence developmentally poised genes until they are ready to be expressed. H2A.Z, an H2A variant, has a state of bivalency that poises genes for inducible expression. H2A.Z links bivalent chromatin to DNA hypomethylation since these poised genes show broad H2A.Z binding and low DNA methylation over their gene bodies.”
In Dup15q brains and cell cultures, the increase in UBE3A led to a decrease of RING1B. This changed levels of H2A.Z and its post-translational modifications.
LaSalle concludes, “It is interesting that the genes affected are transcriptionally plastic and have a unique chromatin signature. It shows something is different about them and that they need to be environmentally responsive in early life. During early development, these genes are located in partially methylated domains (PMDs). PMDs act as a ‘storage box’ for neurodevelopmental genes during early development, which keeps them repressed but poised for brain specific expression during later development.”
Go unwrap the rest of the storage box over at Cell Reports, December 2016